Cryo-Electron Microscopy

What it is, How it Works, Pros and Cons

What is Cryo-Electron Microscopy?

Essentially, Cryo-electron microscopy (Cryo-EM) is a
type of transmission electron microscopy that allows for the specimen of
interest to be viewed at cryogenic temperatures. Following years of improvement,
the cryo-electron microscope has become a valuable tool for viewing and studying the
structures of various biological molecules.

*Transmission Electron Microscopy (TEM) refers
to a technique where the image (of specimen) is formed by directing a high
energy electron beam at a thin sample

While cryo-electron microscopy encompasses a
number of experimental methods (imaging intact tissue sections, imaging plunge frozen
cells and virus etc), these methods are based on the principle of imaging
radiation-sensitive specimens in a transmission electron microscope.

Understanding the Significance of Electrons (Electrons vs. Photons)

Photons are packets of energy (basic particles
of light) therefore, everything that one sees with their own eyes is due to the
fact that these particles reflect off the physical objects we perceive and into our eyes. However, because some of the objects (of specimen in this case)
are too small compared to the wavelength of photons, they are unable to
interact making it impossible to view them.

When it comes to the
wavelength of electrons, it is small enough to interact with the objects making
it possible to observe them. Therefore, electrons become more suitable for the
purposes of observing the small components of cells as well as a variety of
molecular structures.

Transmission Electron Microscope

There are two major types of electron microscopes; the scanning electron microscope and the transmission electron microscope. However, because of the fact that the transmission electron microscopes offer higher resolution compared to the scanning electron microscopes, they are preferred when it comes to structural biology.

The following are some of the major parts of TEMs:

The Electron Column - This is the part of the microscope that contain the electron source (produces electron beam), lenses that focus the electron beam as well as the imaging system where the image is projected

The Vacuum System - This is a component of the microscope that helps minimize collision frequency of electrons with gas atoms. As such, it enhances the efficiency of the electron gun

How the Cryo-EM Works

Transmission electron microscopes use the same
working principle as the ordinary light microscope. However, rather than using
the limited wavelength of light, electrons with much lower wavelengths are used
as the source of light.

For the TEM, there are two types of electron sources that
are commonly used. These include the thermionic electron guns and the field
emission guns. Whereas electrons are emitted from such heated filaments as bent
tungsten or sharp lanthanum hexaboride crystal in thermionic electron guns,
they are emitted from a sharp, pointed cathode by a string electric field in
field emission guns.

For TEM, the electron gun uses electronic coils as well as
high voltages to accelerate the electrons to extremely high speeds (thus shorter
waves). The electrons then travel through the anode, an aperture and into the
vacuum tube. Unlike the light microscope, the TEM has in place electromagnetic
lenses that bend the electron beam though the Lorentz force. The lenses also
direct the beam through the tube and onto the specimen.

Basically, the transmission electron microscope
can be said to have three important systems. These include:

The Electron Gun - The electron gun is the
part of the microscope that is responsible for producing the electron beam.
Here, the condenser system is responsible for focusing the electron beam on to
the specimen sample.

Image Producing System - This is composed of the objective
lens, intermediate and projector lenses as well as a movable stage. The lenses
are also involved in the focusing of electrons, which helps form the magnified
image.

Image Recording System - This part of the
microscope is for most part composed of a fluorescent screen that helps in
producing an image that can be seen with the eye. Here, a digital camera also
helps capture the images for documentation.

Sample

In Cryo-Electron Microscopy the sample under observation is
usually frozen (frozen-hydrated) for preservation purposes. Here, a very thin
slide of the specimen may be rapidly plunged into a liquid ethane bath and
viewed in their natural state. Solvents like water or a salt solution is used
to ensure that the sample remains stable.

* The solvent water around the specimen is
frozen in place when the sample is plunged into the cold medium thus
cryogenically preserving and protecting the specimen.

Here, it is very important that the process of
freezing the sample is very quick. This prevents the frozen water around the
specimen sample from forming cubic ice. In the event that ice is formed,
it readily absorbs the electron beam, which in turn obscures the sample. For this reason,
it is essential that the sample be plunged in to the cooling liquid rapidly so
that the water only freezes around the specimen for clear images.

* While liquid nitrogen can also be used for the
freezing process, ethane is used instead given that it has higher heat capacity and is also
liquid at temperatures slightly above that of liquid nitrogen. As such, it is
sufficiently cold to freeze water rapidly and appropriately without boiling
off.

Sample Preparation

Fixation - This is the first
and most important step of sample preparation for this technique. Here, the
structure of the sample (tissue, cells etc.) has to remain as close to its
natural form as possible.

The sample is then
dehydrated using acetone/ethanol, passed through another transition solvent
(such as propylene oxide) and ultimately infiltrated and embedded in such
liquid resins as epoxy and LR white resin.

This is followed by
sectioning of the sample. Sectioning if performed through ultramicrotomy to
obtain sections of between 50 and 70 nm in thickness

Once collected on a metal
mesh grid, the specimen can then be stained using electron dense stains

To observe macromolecules in their native
hydrated state, the sample is simply embedded in vitrified water, frozen
hydrated as directly visualized on the microscope. Here, no stains are used
given that the surrounding buffer allows for enough contrast to observe the
specimen.

* Here, it is worth noting that different samples
have different protocols. For this reason, it is important to confirm the right
procedure for specific specimen.

Some of the main mounting techniques used in
Cryo-EM include:

Surface Mounting - used mostly for leaf specimens where the specimen is laid
on top of the mounting media

Edge Mounting - Used for edge
observation and fracture, edge mounting technique involves placing the specimen
on its edge in a machine slot secured with a mounting media

Film Emulsion Mounting - This is used particularly in the event that the specimen is too small and can be obscured by the
Tissue-Tek mounting media. Here, the specimen is laid on the surface allowing
it to adhere to the film surface

Pros and Cons of Cryo-Electron Microscopy

Pros

Small Samples

One of the biggest advantages of cryo-electron
microscopy is that very small samples are actually required for the
determination of its structure. Compared to other microscopy techniques, cry-electron microscopy still produces good images (as long as the sample is
in good condition).

A good example of this is with single-particle
reconstruction which only requires 105 particles in order to
reconstruct a near-atomic-resolution structure. Here, quality results can be
expected as long as the sample of interest has good biochemical properties as
well as high conformational homogeneity.

As for such samples as viruses which
have high symmetry, only 104 particles would be required. On the
other hand, a cryo-electron microscope specimen only requires between 3 and 5 ul
of protein solution at a concentration of 1.2 to 1 umol L-1 which is in marked
contrast compared to larger samples used in other techniques like crystallography
and NMR spectroscopy.

Physiological State

The other significant advantage of this
microscopy technique is that fixation of the sample involves rapid freezing and
actual fixation in vitreous ice in order to preserve their hydrated state.
Here, one gets to view a sample with structural information that to a large
extent reflect the state of the sample before it was frozen.

With Cryo-EM, the
sample under observation is in solution and does not come in to contact with
any adhering surface. As a result, the shape observed is also the true shape
(state) of the molecules given that the shape is not affected through attachments
which can result in flattening.

Samples

This technique has an advantage in that it can
be used to view and characterize a wide range of samples. Using cryo-electron
microscopy, it becomes possible to view cells, cell organelles as well as
macromolecules complexes of well over 500 kD.

Recently, Cryo-Electron Microscopy was also used
to determine high resolution structures of 200kD proteins, which was a great
achievement in the world of microscopy. Therefore, apart from only requiring
small samples, this technique can serve to view study and characterize a wide
range of specimen without the need to acquire various other devices.

Inhomogeneous Samples

Cryo-EM offers a great advantage in that it
provides high magnification allowing for the specimen to be viewed and studied
closely. Moreover, it offers a significant advantage in that through the direct
acquisition of the images; the specimen can be statistically analyzed allowing
for the reconstruction of the structural information.

Here, it becomes possible
to classify different possible molecular structures from similar samples to
other molecules with different conformations and compositions. Furthermore, the
classification of a large number of single molecular structures also provides
statistical distribution of different states.

Some of the other benefits of Cryo-Electron Microscopy include:

Stains are not necessary
which means that the specimen is not distorted through the use of stains and
other dyes. Given that low dose methods are commonly used, the electron beam
does not cause much damage to the specimen.

It is possible to
distinguish between nucleic acids, proteins and lipids.

It is possible to control
the chemical environment, which in turn allows for effective examination of
different functional states of different types of molecules.

Cons

Very low signal to noise ratio - This is one of the
biggest disadvantages with Cryo-EM. For biological macromolecules, the main
building blocks include carbon, hydrogen, oxygen and nitrogen. Given that
electron absorption of these molecules is very low, then there is low contrast
of the resulting images which makes it difficult to detect features of a given
sample when viewing a few samples.

Although Cryo-EM offers great overall
magnification, the issues with image contrast may make it difficult for some
users (particularly new users) to distinguish between what they are viewing.

Tilt Imaging - With Cryo-EM, it is not easy to obtain images
from a tilted specimen due to the cross section of the frozen sample. Here, the
cross section of the ice is usually too thick making it difficult to obtain good
images. For this reason, users have to ensure that the specimen is not tilted
in order to obtain better images.

Here, this technique requires that
the user take every step seriously or risk starting all over again. When
imaging a tilted frozen sample, there is also another disadvantage of more
widespread charging.

It is important to ensure that cubic ice is not
formed during freezing. This is due to the fact that they absorb electrons
making the sample worthless. As mentioned, this technique is very sensitive (particularly
with sample preparation) which can be frustrating for some users.

Some of the other challenges with cryo-electron
microscopy include:

It takes time to generate
the sample

Fully hydrated specimen
have been shown to be electron-beam sensitive

It is costly

Conclusion

While it has its challenges, cryo-electron
microscopy has proved to be a valuable tool in various biological fields
including botany, biotechnology and zoology among others. It addition, it is
also being increasingly used in other industries such as pharmaceuticals,
healthcare as well as cosmetics to name a few.

Because it can overcome a number
of limitations that face other microscope techniques like light microscopy and
scanning electron microscopy, Cryo-EM has allowed scientists and technicians to
observe and study a wide range of molecules and their structures.

With
improvements in the few coming years, the problems of Cryo-EM will corrected to
make it one of the best tools in biology, medicine and research.

Given that DNA molecules are found inside the cells, they are too small to be seen with the naked eye. While it is possible to see the nucleus (containing DNA) using a light microscope, DNA strands/th…

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The material on this page is not medical advice and is not to be used for diagnosis or treatment. Although care has been taken when preparing this page, its accuracy cannot be guaranteed. Scientific understanding changes over time.